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,1
* Houston VA Medical Center and the Departments of Medicine and
Pharmacology, Baylor College of Medicine, Houston, Texas 77030
Abstract
Vascular smooth muscle cells (SMCs) generate carbon monoxide (CO) from the degradation of heme by the enzyme heme oxygenase. Because recent studies indicate that CO influences the properties of vascular SMCs, we examined whether this diatomic gas regulates apoptosis in vascular SMCs. Treatment of cultured rat aortic SMCs with a cytokine cocktail consisting of interleukin-1ß (5 ng/ml), tumor necrosis factor-
(20 ng/ml), and interferon-
(200 U/ml) for 48 hr stimulated apoptosis, as demonstrated by DNA laddering, caspase-3 activation, and annexin V staining. However, the exogenous addition of CO (200 ppm) completely blocked cytokine-mediated apoptosis. The antiapoptotic action of CO was partially reversed by the soluble guanylate cyclase inhibitor, H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (10 µM). In contrast, the p38 mitogen-activated protein kinase inhibitor, SB203580 (10 µM), had no effect on SMC apoptosis. These findings indicate that CO is a potent inhibitor of vascular SMC apoptosis and that it blocks apoptosis, in part, by activating the cGMP signaling pathway. The ability of CO to inhibit vascular SMC apoptosis may play a critical role in attenuating lesion formation at sites of arterial damage.
Key Words: cell death • circulation • cell cycle
Carbon monoxide (CO) is an endogenously generated gas that is synthesized principally from the catabolism of heme by the enzyme heme oxygenase (HO)(1). Recent studies indicate that HO-derived CO exerts important biological functions in the circulation. Exogenously administered CO relaxes blood vessels isolated from different vascular sources or animal species (2, 3). Moreover, the endogenous release of CO dilates blood vessels from various vascular beds, suggesting an important vasodepressor function for this gas (2, 3). The HO-catalyzed formation of CO from vascular cells also promotes blood fluidity by inhibiting the aggregation of platelets (4). In addition, CO blocks the synthesis of growth factors from endothelial cells and directly inhibits vascular smooth muscle cell (SMC) proliferation (5–7). These vascular actions of CO are predominantly mediated via the activation of soluble guanylate cyclase and the consequent rise in intracellular guanosine 3',5'-cyclic monophosphate (cGMP) levels (2–6).
Apoptosis, or programmed cell death, plays a fundamental role during vasculogenesis and is associated with numerous vascular disorders, including atherosclerosis, hypertension, and postangioplasty restenosis (8–10). Because SMC apoptosis may contribute to the remodeling response after arterial injury, we determined the effect of CO on vascular SMC apoptosis.
Materials and Methods
Cell Culture and Materials.
Vascular SMCs were isolated by elastase and collagenase digestion of rat thoracic aortas and characterized by immunological and morphological criteria (11). Cells were serially cultured in minimum essential medium containing 10% serum, 5.6 mM glucose, 2 mM L-glutamine, and 100 units of penicillin, streptomycin, and neomycin. Recombinant mouse tumor necrosis factor- and interferon- were purchased from Genzyme (Cambridge, MA); recombinant mouse interleukin-1ß was from R&D Systems (Minneapolis, MN); 1H-[1,2,4]oxadiazolo[4,3-]quinoxalin-1-one (ODQ) and SB203580 were from Calbiochem-Novabiochem Corporation (La Jolla, CA); all other reagents were from Sigma Chemical Company (St. Louis, MO).
Apoptosis.
Apoptosis was monitored by measuring DNA fragmentation on 2% agarose gels, caspase-3 activation, and the movement of phosphatidylserine from the internal to the external surface of the cell membrane, as previously described (12).
CO Exposure.
SMCs were exposed to CO via a previously described environmental chamber (13). Gas from stock tanks containing 1% CO or 5% CO2 in air were mixed in a stainless steel mixing cylinder prior to delivery into the chamber. Flow into the humidified 37°C chamber was at 1 liter/min and CO levels were constantly monitored by electrochemical detection using a CO analyzer (Interscan, Chatsworth, CA).
Statistics.
Results are expressed as the means ± SEM. Statistical analysis was performed with the use of a Student two-tailed t test and P < 0.05 was considered statistically significant.
Results
Treatment of vascular SMCs with a cytokine combination consisting of interleukin-1ß (5 ng/ml), tumor necrosis factor- (20 ng/ml), and interferon- (200 U/ml) for 48 hr induced apoptosis. Although DNA fragmentation was absent in control cells, pronounced DNA laddering was observed in cytokine-treated SMCs (Fig. 1A
). In addition, the cytokine mixture evoked an approximate 2-fold increase in caspase-3 activity after 48 hr of exposure (Fig. 1B). However, exposure of vascular SMCs to CO (200 ppm) inhibited cytokine-mediated DNA laddering and caspase-3 activation (Fig. 1A and B). The antiapoptotic action of CO was also confirmed by measuring the presence of phosphatidylserine on the outer cell membrane. Figure 2 shows representative annexin V binding experiments that quantifies the extent of apoptosis. In both control and CO (200 ppm)-treated SMCs, over 90% of the cells are healthy (Fig. 2A and B). However, after 48 hr of cytokine treatment approximately 60% of the cells are apoptotic (Fig. 2C). Remarkably, this is completely reversed by CO (200 ppm; Fig. 2D).
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). In the absence of CO, ODQ had no effect on DNA laddering (data not shown). Since a recent study (17) demonstrated that p38 mitogen-activated protein kinase (MAPK) mediates the antiapoptotic action of CO in the vascular endothelium, we also determined the involvement of this kinase. However, incubation of SMC with the p38 MAPK inhibitor, SB203580 (10 µM)(17), did not reverse the inhibition of DNA laddering by CO in cytokine-treated cells (Fig. 3) or potentiate the reversal by ODQ (data not shown).
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In the present study we identified CO as a potent inhibitor of vascular SMC apoptosis. Incubation of vascular SMCs with a combination of inflammatory cytokines stimulates apoptosis, as reflected by DNA laddering, caspase-3 activation, and positive annexin V binding. However, the exogenous administration of CO (200 ppm) completely blocks the apoptotic response. This concentration of CO has been shown to be similar to that produced by HO activity in cultured cells and represents a physiologically relevant concentration (13, 17). Our finding that CO blocks apoptosis in vascular SMCs is consistent with recent studies using murine fibroblasts and bovine aortic endothelial cells but it contrasts with other reports employing murine thymocytes and bovine pulmonary endothelium (17–20). The reasons for these divergent results are not known but may reflect differences in the dose and/or duration of CO exposure as well as cell-specific responses to CO.
The mechanism by which CO inhibits SMC apoptosis involves the activation of soluble guanylate cyclase since the soluble guanylate cyclase inhibitor ODQ reverses the antiapoptotic effect of CO. However, other mechanisms are likely to be involved since soluble guanylate cyclase inhibition does not fully reverse the action of CO. Interestingly, the activation of p38 MAPK does not appear to be involved in the antiapoptic effect of CO, since the p38 MAPK inhibitor, SB203580, has no effect on SMC apoptosis. This result contrasts with findings in endothelial cells where CO prevents apoptosis by activating p38 MAPK and indicates that discrete signaling pathways mediate the inhibition of apoptosis by CO in vascular cells (17).
The capacity of CO to block vascular SMC apoptosis may play a critical pathophysiological role in the circulation. Apoptosis has been implicated in plaque progression through the development of an acellular lipid necrotic core (21). SMC apoptosis has also been detected in the vulnerable shoulder regions of plaques suggesting that it may contribute to plaque rupture (8). Furthermore, exposure of phosphatidylserine residues at the surface of apoptotic SMCs enhances their procoagulant potential by promoting the generation of thrombin (22). These findings suggest that CO may serve to limit plaque progression and erosion, and thrombosis. The potential importance of the HO/CO pathway in maintaining vascular homeostasis is supported by studies showing that induction of HO activity suppresses lesion formation in various animal models of atherosclerosis and prevents thrombosis during hypoxia and ischemia-reperfusion injury (23–26). Thus, strategies aimed at delivering CO to sites of arterial damage may represent a novel therapeutic approach in ameliorating vascular disease.
Footnotes
This work was supported in part by the National Heart, Lung, and Blood Institute Grants HL59976, HL36045, and HL62467, and by a grant from the American Heart Association. W. Durante is an Established Investigator of the American Heart Association.
1 To whom requests for reprints should be addressed at VA Medical Center Building 109, Room 130, 2002 Holcombe Blvd., Houston, TX 77030. E-mail: wdurante{at}bcm.tmc.edu 
References
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